There has recently been an alarming increase in infections due to bacteria resistant to all available antibiotics. Tetracycline resistance is typically mediated by efflux pumps; tetracycline derivatives which inhibit them may be used in combination with existing tetracyclines to treat infections by resistant organisms. Flow cytometric methods will be developed to facilitate measurement of tetracycline uptake and efflux in bacteria, and detection of effects of tetracyclines and daptomycin on membrane potential, pH, and permeability. Another method of potentiating existing antibiotics is suggested by the finding that bacteria treated with sublethal doses of some antibiotics become transiently permeable to dyes that normally do not penetrate the membranes of either bacterial or mammalian cells. The chemical modifications used to make dyes impermeant can be applied to various toxic agents; these modified agents should be nontoxic to host cells, but kill transiently permeabilized bacteria. This hypothesis, which may provide a novel and general therapeutic approach to bacterial infections, will be tested using fluorescent nucleic acid binding compounds with known permeant and impermeant characters; antibiotic-sensitive and -resistant Staphylococcus and Enterococcus species and Escherichia coli will be examined. PROPOSED COMMERCIAL APPLICATION: There is a substantial market in drug companies and research institutions for assays and related instrumentation which facilitate rapid development of new antimicrobial agents. Moreover, drugs or combinations which improved treatment of infections due to even a few antibiotic-resistant bacterial species would capture a significant share of a huge market for antibacterial agents.